The phosphatase enzyme catalyzes the hydrolysis of phosphate groups, removing phosphates from various substrates. Within the family of phosphatase enzymes there exist members that are particularly active under alkaline pH conditions. Such phosphatases are commonly referred to as alkaline phosphatases. Still further, within this sub-family of alkaline phosphatases there are members that are particularly unstable at temperatures above 37° C. Such phosphatases are referred to generally as heat labile alkaline phosphatases.
Alkaline phosphatases (E.C. 3.1.3.1) are also commonly referred to as alkaline phosphomonoesterase, phosphomonoesterase or glycerophosphatase. These enzymes are orthophosphoric-monoester phosphohydrolases with enzyme activity optima at alkaline conditions. Examples of alkaline phosphatase substrates are deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and ribo-, as well as deoxyribo-, nucleoside triphosphates, alkaloids and phosphate-containing proteins or polypeptides. The hydrolysis reaction catalyzed by phosphatase enzymes yields an alcohol and an orthophosphate. In other words, alkaline phosphatases dephosphorylate DNA, RNA, rNTPs and dNTPs. Dephosphorylation of protein by various alkaline phosphatases have also been reported. Alkaline phosphatases may be found in organisms ranging from bacteria to humans. Complex organisms may contain tissue-specific and non-tissue specific alkaline phosphatases. Alkaline phosphatases generally range in size from 15 kDa to 170 kDa. Some of these proteins are bound or “anchored” to cellular membranes. Alkaline phosphatases may require various co-factors for optimal activity, such as metal cations, like Mg2+, Zn2+ or Co2+.
Alkaline phosphatases are often used in molecular biology applications, such as: 1) dephosphorylation of vector DNA after restriction enzyme digestions to minimize self-ligation of the cloning vector, thus favoring ligation of the insert to the vector and creating a recombinant construct, 2) dephosphorylation of dNTPs after PCR amplifications, occasionally in combined use with a single-strand exonuclease that hydrolyses primers to dNTPs, to omit the need of further clean-up before direct DNA sequencing of PCR products or SNP genotyping, or 3) dephosphorylation of DNA ends for subsequent labeling with 32P using [γ-32P]NTP and T4 polynucleotide kinase. The aforementioned alkaline phosphatase reactions are intermediate steps in typical DNA analysis processes. Alkaline phosphatases are also commonly used in reporter systems, such as in enzyme-linked immunosorbent assays (ELISA), in gene-fusion or gene-delivery systems, or in conjugation to oligonucleotides used as hybridization probes.
At least three alkaline phosphatase enzymes are commercially available and commonly used in molecular biology applications, including: i) calf intestinal alkaline phosphatase (CIP), ii) shrimp alkaline phosphatase (SAP) from the arctic shrimp Pandalus borealis, and iii) bacterial alkaline phosphatase (BAP), isolated from Escherichia coli. Bacterial Alkaline Phosphatase dephosphorylates all types of DNA ends but is difficult to inactivate because it is very resistant to heat and detergents. (See, Sambrook et al., Molecular Cloning, A Laboratory Manual, §1.53-1.72, 1989). CIP is also widely used in molecular biology techniques (for instance, see U.S. Pat. Nos. 5,773,226 and 5,707,853) but requires the use of Proteinase K treatment followed by phenol:chloroform extractions, or a heat step followed by phenol:chloroform extractions, to remove the enzyme when the reaction is completed.
A genetically engineered temperature sensitive BAP mutant has been reported. (See, Shandilya et al., 1995, Focus, 17 (3):93-95). This mutant enzyme (TsAP), sold by LifeTechnologies, Inc., is inactivated (95% or more) by heat (65° C. for 15 minutes) in the presence of EDTA.
Psychrophiles, or cryophiles, are extremophilic organisms that are capable of growth and reproduction in cold temperatures. In recent years, thermolabile alkaline phosphatases have been developed. Among them is the heat labile phosphatase from the psychrophilic strain TAB5 which was discovered in Antarctica and is referred to as Thermolabile Antarctic phosphatase (TAP). (See, Rina et al., Eur. J. Biochem. 267:1230-1238, 2000, and U.S. Pat. No. 7,319,014). TAP is heat labile and has a high specific activity; but does not possess significant dephosphorylation activity of dNTPs after PCR amplification.
A psychrophilic alkaline phosphatase (PAP), developed in Japan and isolated from the Shewanella sp. SIB 1 has been reported and is a cold-active alkaline phosphatase. (See, Japanese Patent No. 2001-172653). It has been reported that PAP has high specific activity at low temperature (Ishida et al., 1998, Biosci. Biotechnol. Biochem., 62, 2246-2250). It is not known whether PAP is active in dephosphorylating dNTPs after PCR amplification.
Two additional heat-labile alkaline phosphatases from a psychrophilic microorganism have been purified and characterized. (See, de Prada et al., 1997, Appl. Env. Microbiol., 63 (7): 2928-2931). However, no specific activity (units/mg protein) and no primary structures have been reported for these enzymes.
A second cold-adapted alkaline phosphatase, this one from atlantic cod, was isolated and characterized. (See, Asgeirsson et al., 1995, Comp. Biochem. Physiol., 110B (2): 315-329). The cod alkaline phosphatase exhibited thermolability similar to SAP. No primary structure of the protein/gene has been provided. Further, trout fish alkaline phosphatase isozymes have been isolated. (See, Whitmore et al., 1972, J. Exp. Zool., 182: 59-68). Additionally, shrimp from the warm water region near Taiwan express several alkaline phosphatases. (See, Lee et al., 1991, Comp. Biochem. Physiol., 99B (4): 845-850).
SAP, isolated from the arctic shrimp Pandalus borealis, was found in the processing wastewater from the shrimp industry. (See, Olsen et al., 1990, Process Biochem., 25:67-68). This enzyme was later identified as originating from shrimp hepatopancreas. (See, Olsen et al., 1991, Comp. Biochem. Physiol., 99B (4):755-761). This alkaline phosphatase possesses maximum enzyme activity at about 40° C., whereas CIP possesses maximal activity at about 45° C. Although the temperature for maximum activity is close to 40° C., SAP looses activity when pre-incubated for a period of 15 minutes at temperatures above 37° C. SAP has been reported to loose 95% of activity if pre-incubated at 65° C. for 15 minutes. In comparison, after similar heat-treatments, CIP retains 40% activity.
As noted above, commercial SAP is obtained from wastewater generated by the shrimp industry. Freshly collected shrimp are first frozen in large blocks. Then, the frozen shrimp are thawed by re-circulated cold water. During the process of freezing and thawing, the hepatopancreas of the shrimp are ruptured and the contents thereof are released into the circulating water. This wastewater is then concentrated and several protein purification steps are employed to purify SAP.
SAP is frequently used to dephosphorylate cloning vectors prior to ligation reactions, and to treat PCR amplification product-mixtures prior to DNA sequencing reactions, as described in U.S. Pat. Nos. 5,741,676, 5,756,285, 6,379,940 and 6,387,634.
Production efficiency of SAP suffers from varying quality of the wastewater. Variation in the yield quality of SAP from this natural source stems mainly from the natural seasonal variation of enzyme production in the shrimp, handling of the shrimp source prior to or during freezing, and handling of the shrimp or water during or after the thawing process. Further, there is growing concern about the future availability of shrimp collection wastewater. As a natural resource, shrimp may be depleted through over-fishing or other acts of nature. Changes in the shrimp industry, i.e. single-freezing, and processing of the shrimp, may also eliminate the wastewater source. (See, U.S. Pat. No. 7,323,325).
Thus, there is a demand for a recombinant SAP product which is sustainable. Recombinant products may be preferred in molecular biology applications where product purity is important, e.g. in the production of DNA based therapeutics or in forensic science, and where strict standardization is required. There is therefore a desire in the field for a synthetic or recombinant source of alkaline phosphatase which is produced in a uniform and pure fashion.
Recently, a recombinant SAP has been developed. (See, U.S. Pat. No. 7,323,325). However, recombinant SAP is not presently commercially available.
The molecular biology field, as well as other similar fields using alkaline phosphatases, would significantly benefit from a source of isolated, high-quality, recombinant heat labile alkaline phosphatase enzyme. However, such commercial availability encompassing all of these attributes is as yet unrealized.
Psychrophilic organisms have successfully adapted to various low temperature environments such as cold ocean waters. Thermal compensation of cold adapted enzymes found in such organisms is realized through improved turnover number and catalytic efficiency, and a highly flexible structure. (See, Feller et al., Cell. Mol. Life Sci., 53:830-841, 1997). In such environments, organisms produce enzymes with increased catalytic efficiencies, generally at the expense of thermal stability due to fewer non-covalent stabilizing interactions. (See, Hauksson et al., Enzym. Micrbiol. Tech., 27:66-73, 2000).
Colwellia psychrerythraea is a non-pathogenic, obligate psychrophile and Gram-negative bacteria. C. psychrerythraea is a member of the proteobacteria phylum, class gammaproteobacteria. This bacterium is rod-shaped, red in pigment, possesses flagella and can be found in cold marine environments such as the Arctic and Antarctic sea ice. Strain 34H, in particular, was isolated from Arctic marine sediments. Strain 34H of C. psychrerythraea has a growth temperature range of from −1° C. to 10° C. Optimal growth appears at 8° C., with maximum cell yield occurring at the subzero temperature of −1° C. Cells are able to survive in temperatures as low as −10° C. Growth can occur under deep sea pressure as well.
Alkaline phosphatases are in general difficult to express in E. coli and heat labile family members tend to present an even greater challenge due to their low thermal stability. Many problems may occur during expression of the protein and many hurdles typically present themselves to obtaining purified, active protein. One problem is that expressed protein may be abundant, but inactive, i.e. expressed but conglomerated in unusable inclusion bodies in the bacterial host. The degree of inactivity of isolated recombinant protein may be related to growth conditions (growth and induction temperature, media composition, induction time).
An expression system, media composition and growth conditions are disclosed herein which are capable of yielding sufficient quantities of active recombinant C. psychrerythraea heat labile alkaline phosphatase (CAP). The present methods enable production of sufficient quantities of CAP protein to be commercialized, as described in general, below. Disclosed are methods of over-expression and purification of the recombinant alkaline phosphatase and mutants thereof. Particularly, disclosed are methods of over-expressing and purifying commercially useful quantities of active recombinant heat labile alkaline phosphatase fusion enzymes from C. psychrerythraea, wherein the fusion enzymes comprise one or more heterologous sequences. The disclosed C. psychrerythraea heat labile alkaline phosphatase has properties similar to shrimp alkaline phosphatase and can be substituted for shrimp alkaline phosphatase in assays involving the same.